Reagent and procedure for the detection of pathogens, especially spirochetes from body fluids

ABSTRACT

A reagent for microscopy-based detection of pathogens, especially spirochetes, from body fluids characterized by that containing the following ingredients: tetracain (125-200 mg/l), mannit (1500-2000 mg/l), EGTA (etilene bis[oxyetiline-nitrilo]-tetraacetate), 0.76-0.114 mg/l magnesium-chloride (preferably in amount of 0.10 mg/l), caffeine-sodium-benzoate (2000-4000 mg/l), glucose (1800-2200 mg/l), glycerol (75-105 mg/l), optimally tri-sodium-citrate (preferable in an amount of 10000 mg/l), Hoechst 33342 dye (1.11 mg/l), if required 20 to 40 ml of distilled water and RPMI 1640 culture media to make 100 ml. Also, a method for detecting pathogens.

This application claims priority of the earlier filing date, under 35U.S.C. 119, of PCT International Application No. PCT/HU00/00013, filedon Feb. 16, 2000, published in English.

BACKGROUND OF THE INVENTION

The invention is a reagent and a microscopy-based procedure for thedetection of pathogens, especially spirochetes, from body fluids.

Provided that sterility is observed, the reagent according to theinvention makes it possible to examine human body fluid samples under adark-field microscope without staining.

As early as in 1683, the Dutch scientist, Anthonij van Leeuwenhoekdiscovered the spirochetes through his microscope and informed theBritish Royal Society about his discovery. Two centuries later, JózsefFodor was the first to discover that the blood of healthy individualsdoes not contain germs.

In comparison to serological and PCR techniques, the independence of themorphological examination (microscopy) of antigenicity and other changes(which—among spirochetes—is most typical of Borreliae) as well as of theemergence of new subspecies is still considered an advantage. Thegenetic make-up and the phenotype of these pathogens can even changeduring the illness of a single person, and some of these changes may notbe detectable using the current techniques. After excluding infectionswith incompatible clinical presentations and higher local probabilities,morphological identification of the causative agent is diagnostic.

Despite the fact that the existence of spirilla was unequivocallydemonstrated with the use of the microscope, even the well-known andwidely recognized studies of Leeuwenhoek were forgotten—only to bere-discovered centuries later.

Indeed, conventional staining can hardly make these extremely thin andlong pathogens visible; the tedious and most sophisticated technique ofsilver impregnation is still the only way to detect them in smears andslides. Cultivation is still problematic.

Since 1909, however, dark-field microscopy has made it possible tofirmly diagnose spirochetoses from native, not fixed slides before theappearance of antibodies, provided the samples are taken from a patientwho shows certain well-defined symptoms.

This means that the diagnosis can be made at an early stage, whentreatment is expected to be most effective.

It was microscopy that proved that from the portal of entry, pathogensreach all organs (including the CNS) via the bloodstream and thelymphatic system.

The laboratory diagnosis of borrelioses with different clinicalpresentations is based primarily on the detection of spirochetes fromblood samples. This is easily accomplished in recurrent fever becausenormally, there is a large number of B. recurrentis present. Besides,other morphological properties of this pathogen (shown in the tableenclosed) and the fact that this pathogen is easy to stain also make itsdetection easier. There are mild cases, however, when the symptomssuggest the diagnosis of recurrent fever but the cell count is too lowfor the conventional methods to detect the causative agent. To solvethis problem, the technique of microhematocrit concentration (doublecentrifugation of blood samples) has been used since 1972. Microscopy issuperior in that the test result is not affected by the changingantigenicity of Borreliae. [Goldsmid, J M. Mohamed: The use ofMicrohematrocit Technic for the Recovery of Borrelia duttonii from theBlood, Am. J. Clin. Pathol. 58:165-169 (1972)].

Sample concentration has also been used to enhance conventionalmicroscopy in parasitology. This way, pathogens can be detected moreefficiently.

The diagnosis of Lyme borreliosis and the identification of itscausative agent emerged as a new problem.

It is known that, in the seventies there was an outbreak of arthritiscases among the children who lived around the town of Lyme, Conn., USA.Ticks were soon identified as the vectors but the causative agent of themysterious Lyme disease was not identified until much later.

After the unsuccessful investigations using top technologies, WillyBurgdorfer discovered the causative agent with a microscope andidentified it as a new spirochete. This pathogen is referred to asBorrelia burgdorferi sensu lato [(Burgdorfer, W, Barbour, A. G., Hayes,S., F., Benach, J. L, Grunwaldt, E., Davis, J. P: Lyme Disease—atick-borne spirochetosis? Science, 216(4552): 1317-9, Jun. 18, 1982)].

As mentioned above, morphological examination has long been included inthe laboratory diagnosis of spirochetoses. If dark-field microscopy isemployed, concentrated fluid samples do not need to be stained, whichmeans that the long and thin spirochetes are not washed off the slides,which, in turn, increases sensitivity.

Soon after the introduction of this technique, which has now been usedfor decades, it was noted that pseudospirochetes (also known as myeloidfigures), which are formed mainly during the degradation of red bloodcells, can mislead the examiner whether it is human or animal blood thatis examined. These artifacts are most likely to be present when storedsamples or the stomach contents of blood-sucking insects (louse orticks) are examined for infection. This issue is addressed in thefollowing articles:

Chamber, H.: A new spirochaeta found in human blood; Lancet 1913; 1:1728-1729;

Brecher, G.; Bessis, M: Present status of spiculed red cells and theirrelationship to the discocyte, echinocyte transformation: a criticalreview. Blood 1972; 40: 333-344;

Smith, T F.; Wold, A D.; Fairbanks, V F.; Washington, J A 2nd;Wilkowske, C J.: Pseudospirochetes a cause of erroneous diagnoses ofleptospirosis. Am. J.Clin. Pathol. 1979; 72: 459-63;

Greene,R T.; Walker,R L.; Greene,C E.: Pseudospirochetes in animal bloodbeing cultured for Borrelia burgdoiferi. J.Vet. Diagn.Invest. October1991; 3(4): 350-2;

Mursic,V P.; Wanner,G; Reinhardt,S; Wilske,B; Busch,U; Marget,W:Formation and cultivation of Borrelia burgdorferi spheroplast-L-formvariants. Infection. 1996; 24: 218-26.

At the same time, however, Borrelia burgdorferi sensu lato even hasseveral different morphological patterns in cultivation. Degenerativeforms are also present if antibiotics are added to the culture(Aberer,E; Duray,P H: Morphology of Borrelia burgdorferi: structuralpatterns of cultured borreliae in relation to staining methods.J.Clin.Microbiol. 1991; 29(4): 764-72;

Barbour,A G; Todd,W J; Stoenner,H G: Action of penicillin on Borreliahermsii. Antimicrob.Agents.Chemother. May; 21, 1982(5): 823-9;

Kersten,A; Potschekm,C; Rauch,S; Aberer,E: Effects of penicillin,ceftriaxone, and doxycycline on morphology of Borrelia burgdorferi.Antimicrob.Agenst.Chemother. 1995; 39(5): 1127-33.)

Today the diagnosis of spirochetoses is not problematic, except forborrelioses, which still present a challenge worldwide.

It is the direct identification of Borrelia burgdorferi sensu lato thatcan unambiguously verify Lyme disease. This is a tedious and ineffectiveprocess because the causative agent is difficult to cultivate and thelow cell count of tissue and body fluid samples makes microscopyunworkable. Not even the newer genetic tests are sensitive enough inthis case.

The indirect techniques used in the diagnosis of Lyme borreliosis arediscussed below.

In this approach, host antibodies produced in response to Borreliaburgdorferi sensu lato infection are detected; the idea behind it isthat nothing else but the infection can cause the antibody titers torise. During the first stage of this infection, antibody production isslower than usual. Antibodies do not appear until weeks after theinfection and are only rarely present throughout the whole course of thedisease because titers keep changing and—after some time—they may becomenormal without any intervention. This makes it difficult to define thethreshold titer. There is no clinically applicable threshold that couldmake a clear-cut distinction between those who are infected and thosewho are not. Besides, generation cycles of the causative agent cause afluctuation of the early—IgM type—antibody titers. As far as we know,this is the only disease in which the causative agent blocks theproduction of the more specific and more effective IgG type antibodies,which normally follows the production of IgM. There are even cases ofLyme borreliosis in which only the early (IgM type) antibodies arepresent years after the infection.

What has been said so far affects all antibody assays. That is to say,comparative studies can only compare the sensitivity of the techniquesin question.

Thus, it would be a big mistake to base the laboratory diagnosis of Lymeborreliosis on the traditional evaluation of a single test. Test resultsare sometimes considered non-specific in this case. The chances ofnon-specific reactions are known to be higher in spirochetoses but theycan be avoided with traditional pre-test absorption, which removes thenon-specific antibodies that could give a false reaction. If the testresult is negative, physicians may doubt the validity of the patients'complaints and abandon the possibility of Lyme borreliosis even thoughantibody production may be inadequate or blocked, the technique employedmay not be able to detect all antibodies or the threshold value may notbe set correctly.

At the same, the clinical presentation may be compatible with thediagnosis but traditional techniques may not verify it.

The existence of spirochetoid bodies and the assumption that they areidentical with Borrelia burgdorferi sensu lato is indirectly verified bythe fact that the prevalence of the disease that they cause is quitehigh world-wide and Hungary has an even higher rate of infection. Theresults of a comparative study shown in Table III and FIG. 1 provideindirect and direct evidence, respectively. High prevalence may beexplained by the fact that despite the availability of very potentantibiotics, the treatment of this disease is still difficult.Therefore, it may well be assumed that there is a continuousaccumulation of cases. Mention must be made of spontaneous healing,which is a theoretical possibility but has not been proven, sincerelapse may even occur after 30 years of asymptomatic disease.

As is known, both tick-borne encephalitis and Lyme borreliosis candevelop after the same tick-bite. In Hungary, reliable data is availableregarding the incidence of tick-borne viral encephalitis, which has beenincluded in the national disease surveillance for almost two decades:between 200 and 400 new cases are reported each year. We know whatpercentage of ticks carries the bacterial and viral pathogens. Theincidence of Lyme borreliosis (LB) can then be calculated as follows:${LB} = {\frac{{the}\quad {percentage}\quad {of}\quad {ticks}\quad {carrying}\quad {the}\quad {bacteria}}{{the}\quad {percentage}\quad {of}\quad {ticks}\quad {carrying}\quad {the}\quad {viruses}}*{the}\quad {incidence}\quad {of}\quad {KEC}}$

The percentage of ticks carrying the viruses varies in different partsof the country but on average, 1 of 1000 ticks is infected in Hungary.

Many more ticks—at least 1 of ten—carry the bacteria, however. This istrue of all of Hungary and even Europe; the causative agent of Lymeborreliosis can be detected in at least 1 of 10 ticks collected anywherein Europe.

In short, the possibility of being infected by Borrelia burgdorferi isone hundred times higher than that of tick-borne viral encephalitis(KEC).

This helps us to estimate the incidence of Lyme borreliosis, the exactlaboratory diagnosis of which is yet to be determined.

Based on what has been said so far, the incidence of Lyme borreliosiscan be estimated as follows:${LB} = {\frac{1\text{/}10}{1\text{/}1000}*200}$

The literature and the above formula both predict at least 20,000 newcases in Hungary each year. After tick bites, the viral and bacterialinfections are expected to develop independently while the number ofcases are expected to reflect the ratio of ticks carrying the twopathogens. If one also accepts the validity of the fact that only aninsignificant number of Lyme borreliosis patients were treatedadequately before 1990, then it might be inferred that in Hungary, asmany as maybe one million people (at least 10 percent of the populationtoday) may have become infected with Lyme borreliosis during the pastsixty years.

This makes the diagnosis of Lyme borreliosis much more probable thanthat of other spirochetoses. Therefore, the spirochetoid bodies are mostlikely to be Borrelia burgdorferi sensu lato; this may also be a validstatement for other continents provided that the test result is viewedin the light of the clinical presentation.

Lyme borreliosis can often be diagnosed using conventional laboratorytechniques and can often be cured with antibiotics. However, no reliabletest to identify the seronegative cases of Lyme borreliosis (when theclinical presentation strongly suggests the diagnosis but serologicaltechniques fail to verify the infection) has existed until now.

Lyme borreliosis reinfections are also known to occur due to the factthat—because of antigenicity changes and specific immunosuppression—hostantibodies are unable to provide protection. The shedding of Borreliaburgdorferi sensu lato shown in Figure enclosed may also play a role inthis process. Reinfection can therefore result in disease. Theseronegative form of Lyme borreliosis is quite common.

In short, there is no single diagnostic technique that could verify allcases of Lyme borreliosis at present.

The aim of the invention is suppletory: to work out a technique and areagent which is more suitable for the detection of spirochetes and canreliably verify all clinical forms of Lyme borreliosis provided thatclinical symptoms are also taken into account. When used as a screeningtest, positive results mean that close follow-up is needed. The inventedtechnique can eliminate the disadvantages of other techniques.

The following considerations led to the development of the invention.

In view of the state of the art in diagnostic techniques discussedabove, the problem has been tried to solve by improving themicroscopy-based technique. To achieve this aim it has been to make surethere is a sufficient number of pathogenic spirochetes in the samplesand there are no artifacts.

As noted above, the study of the first cases of Lyme borreliosis made itclear that it is only via the circulation that Borrelia burgdorferisensu lato can disseminate from the site of the tick bite and reachdifferent organs.

In a study made at a university in Vienna, Austria in 1985, ProfessorStanek and his colleagues found that in artificially infected laboratoryanimals bacteremia could be detected using dark-field microscopy as wellas conventional microscopy after Giemsa staining: the Borreliaburgdorferi sensu lato injected subcutaneously appeared in thecirculation and remained detectable continuously. The number of bacteriadetected was changing in seven to eight day cycles. They realized thatthe number of pathogens is changing and that in certain periods of thegeneration cycle spirochetes are more difficult to detect. [(Stanek, G.;Burger, I; Hirschl, A.; Wewalka, G.; Radda, A: Borrelia transfer byticks during their life cycle Studies on laboratory animals. Zbl. Bakt.Mikrobiol. Hyg. A., 263: 29-33: 1986)]

In a paper published in 1998, a research team that had been workingunsuccessfully on the cultivation of Borrelia burgdorferi from blood foryears described a procedure for increasing the yield of blood cultures.They used this procedure to prove the assumption that the reason fortheir previous failure was insufficient sample quantity rather than thelack of pathogens. [(Wormser, G P et al: Improving the Yield of BloodCultures for Patients with Early Lyme Disease, J. Clin. Microbiol.36:296-298, 1998)].

All this supported the theory that dark-field microscopy can be adaptedto the examination of concentrated blood and other body fluid samples.However, it has been realised that until the formation of myeloidfigures is prevented, dark-field microscopy couldn't be put intopractice.

GENERAL DESCRIPTION OF THE INVENTION

As the title suggests, a new reagent has been developed that can slowdown the aging (membrane hardening) of human erythrocytes, leukocytes,platelets and squamous epithelial cells in the samples. It has beenfound that the reagent according to the invention stops the amoeboidmovement of leukocytes and the fragmentation of platelets. Thus, myeloidfigures are not formed. The membrane of accidentally formed myeloidfigures is also hardened. Consequently, they do not even exhibitBrownian movement; they simply float along. The movement and the celldivision of Borrelia burgdorferi sensu lato remained unaffected by theinvented reagent. This is how shedding could be observed, which had onlybeen noted in cell cultures.

DETAILED DESCRIPTION OF THE INVENTION

The essential ingredients of the reagent according to the invention arethe following: tetracain (125-200 mg/l), mannit (1500-2000 mg/l), EGTA(etilene-bis[oxyetiline-nitrilo]-tetraacetate) 0.76-0.114 mg/l,magnesium-chloride (preferably in an amount of 0.60 mg/l),caffeine-sodium-benzoate (2000-4000 mg/l), glucose (19000-22000 mg/l),glycerol (75-105 mg/l), tri-sodium-citrate (preferably in an amount of10000 mg/l), Hoechst 33342 dye (1.00 mg/l,) if required 20 to 40 ml ofdistilled water and RPMI 1640 culture media to make 100 ml.

During the procedure according to the invention, a given amount ofreagent is added to human blood or other body fluid samples. The samplesare then shaken, incubated and stored, if needed, Blood cells, plateletsand pathogenic bacteria are then separated from the rest of the sample,and then the remainder is concentrated and examined under a microscope.

Blood cells, platelets and pathogenic bacteria can be separated withsedimentation, slow centrifugation or filters. Concentration isaccomplished with high-speed centrifugation.

It has been solved by the invention, that a simple and cheap traditionaltechnique can be used to test human body fluid samples too. In ourexperience, it is still possible to detect the pathogenic bacteria ifthere are less than 10 bacteria in a milliliter of centrifuged nativeblood samples. In comparison, the threshold for the detection of Lymeborreliosis with PCR, which is currently considered the most sensitivebut can only be done in specially equipped laboratories, is between 40and 100 germs per ml; besides, as many as possible primers specific todifferent sub-strains should be available.

It should be noted that further morphological, immunocytological andimmunoserological examination of the centrifuged sample treated with thereagent according to the invention is also possible. Furthermore, it canalso be utilized for PCR and cultivation. In the latter cases, filteringis recommended before concentrating the sample.

The analysis of examination data can also provide the basis for a noveltheoretical classification that reflects more aspects of thepathogenesis of Lyme borreliosis.

The technique according to the invention makes it possible to study thecurrent state of pathogenesis and determine the activity of Lymeborreliosis in a given patient. A fast and reliable diagnosis can bemade even when serological tests are negative. The reagent and theprocedure according to the invention provides reliable data to aidtreatment (which is still controversial), monitor treatment effects andpredict relapses before the development of humoral immune response orafter it has been blocked and all this is independent of autoimmuneresponses.

BEST MODE OF CARRYING OUT OF THE INVENTION

The following is a list of examples of the invented reagent, withoutrestricting.

EXAMPLE 1

Composition of the reagent: Hoechst 33342 dye, 1.00 mg/l, tetracain 200mg/l, mannit 2000 mg/l, EGTA 0.76 mg/l, magnesium-chloride (containing 6H₂O molecule), 0.61 mg/l, caffeine-sodium-benzoate 4000 mg/l,tri-sodium-citrate 10000 mg/l, glucose 20000 mg/l, glycerol (87%) 105mg/l, distilled water 30 ml, RPMI 1640 culture to make 100 ml (pH=7.25;μ=2.10)

EXAMPLE 2

The ingredients are essentially the same as in Example 1 but theingredients of the culture media are also listed. The reagent thuscontains the following ingredients:

pH = 7.25 Composition of the Reagent μ = 2.10 1. Arginine 200.00 mg/L 2.Asparagine (H₂O) 56.82 mg/L 3. Asparaginic acid 20.00 mg/L 4. Biotine0.20 mg/L 5. Vitamin B₁₂ 0.005 mg/L 6. Cysteine-disodium 59.00 mg/L 7.EGTA 0.76 mg/L 8. Phenilalanine 15.00 mg/L 9. Phenole Red 5.00 mg/L 10.Folicacid 1.00 mg/L 11. Glycerol (87%) 105.00 mg/L 12. Glucose 22000.00mg/L 13. Glutanic acid 20.00 mg/L 14. Glutamine 300.00 mg/L 15.Glutathione 1.00 mg/L 16. Glycine 10.00 mg/L 17. Histidine 15.00 mg/L18. Hictoxyprolyn 20.00 mg/L 19. Hoechst 33342 1.00 mg/L 20. Inosite35.00 mg/L 21. Isoleucyn 50.00 mg/L 22. Calciumpantotenole 0.25 mg/L 23.Calcium-nitrate (Ca(NO₃)₂, 70.00 mg/L 6*H₂O 24. Calcium-chloride 400.00mg/L 25. Caffeine-sodium-bensoate 4000.00 mg/L 26. Cholin-chlorid 3.00mg/L 27. Leucine 50.00 mg/L 28. Lysine 40.00 mg/L 29. Magnesium-chloride6*H₂O 0.61 mg/L (5 mM) 30. Magnesium-sulphate (7*H₂O) 100.00 mg/L 31.Mannite 2000.00 mg/L 32. Methionine 15.00 mg/L 33. NaCl 6000.00 mg/L 34.NaHCO₃ 2000.00 mg/L 35. Na₂HPO₄ 800.00 mg/L 36. Nicotine-amid 1.00 mg/L37. Para-amino benzoic-acid 1.00 mg/L 38. Proline 20.00 mg/L 39.Pyridoxin 1.00 mg/L 40. Riboflavine 0.20 mg/L 41. Serine 30.00 mg/L 42.Tetracaine 200.00 mg/L 43. Thiamine 1.00 mg/L 44. Thyrosine disodium25.00 mg/L 45. Threonine 20.00 mg/L 46. Trisodium-citrate 10000.00 mg/L47. Trypthofhan 5.00 mg/L 48. Valine 20.00 mg/L 49. Distilled Water ad1000.00 mL

EXAMPLE 3

3.0 milliliters of the invented reagent are added to 7.0 milliliters ofa human blood sample or a maximum of 10.0 milliliters of a human bodyfluid sample. The sample is shaken and incubated at +4 degrees Celsiusfor 120 minutes. The sample is then stored for a maximum of 24 to 72hours. Double centrifugation is then performed at +4 degrees Celsius.First, the sample is centrifuged at 800 g for 10 minutes. Thesupernatant can then be filtered before it is concentrated at 20,000 gfor 20 minutes. A seven milliliter sample yields a concentratedprecipitate of 10 microliters, of which three microliters are examinedunder the microscope.

EXAMPLE 4

The procedure according to the invention is executed applying thereagent of Example 1. Sample sterility is maintained and—after theremoval of antibodies, blood cells and platelets and increasing theconcentration of pathogenic bacteria—culturing and other conventionalnon-serological procedure can be performed more efficiently.

The following is a series of tables respectively shown in FIGS. 3-5.Table 1, shown in FIG. 3, lists the common human infections byspirochetes and their causative agents, Table 2, shown in FIG. 4,demonstrates the differential diagnosis of spirochetoses and Table 3,shown in FIG. 5, deals with comparative studies.

Two figures (FIGS. 1 and 2) about the causative agent and its sheddingare included as attachments.

What is claimed is:
 1. A reagent for microscopy-based detection ofpathogens from body fluids, containing the following ingredients:125-200 mg/l of tetracaine, 1500-2000 mg/l of mannite, 0.76-0.114 mg/lof etilene bis[exyetiline-nitrilo]-tetraacetate, about 0.60 mg/l ofmagnesium-chloride, 2000-4000 mg/l of caffeine-sodium-benzoate,1800-2200 mg/l of glucose, 75-105 mg/l of glycerol, 10000 mg/l oftri-sodium-citrate, 20 to 40 ml of distilled water and a culture media.2. The reagent according to claim 1, characterised in that the pathogensare spirochetes.
 3. The reagent according to claim 1, characterised inthat the ingredients further include 1.00 mg/l of dye.
 4. A procedurefor microscopy-based detection of pathogens from body fluids,characterised in that a given amount of the reagent according to claim1, is added to a sample of human blood as the body fluid, the sample isshaken, incubated, the blood cells and the platelets are then separatedfrom the sample, and the sample is concentrated and examined under amicroscope to detect pathogens which may be contained therein.
 5. Theprocedure according to claim 4, characterised in that the separation ofthe blood cells and platelets from the sample is carried out bysedimentation, slow centrifugation, and high-speed centrifugation. 6.The procedure according to claim 5, characterised in that it is employedin the detection of Borrelia burgdorferi sensu lato.
 7. The procedureaccording to claim 4, characterized in that the sample concentration iscarried out by high-speed centrifugation.
 8. The procedure according toclaim 7, characterised in that it is employed in the detection ofBorrelia burgdorferi sensu lato.
 9. The procedure according to claim 4,characterised in that it is employed in the detection of Borreliaburgdorferi sensu lato.
 10. The procedure according to claim 4,characterised in that the pathogens are spirochetes.
 11. The procedureaccording to claim 4, characterized in that the sample is stored afterbeing incubated, and before the blood cells and the platelets areseparated from the sample.
 12. A method for microscopy-based detectionof pathogens from body fluids, the method comprising the steps of:mixing a given amount of reagent to a sample of blood, the reagentcontaining: 125-200 mg/l of tetracaine, 1500-2000 mg/l of mannite,0.76-0.114 mg/l of etilene bis[exyetiline-nitrilo]-tetraacetate, about0.60 mg/l of magnesium-chloride, 2000-4000 mg/l ofcaffeine-sodium-benzoate, 1800-2200 mg/l of glucose, 75-105 mg/l ofglycerol, 10000 mg/l of tri-sodium-citrate, 20 to 40 ml of distilledwater, and a culture media; incubating the sample; separating bloodcells and platelets from the sample; concentrating the sample; andexamining the sample with a microscope to detect pathogens which may becontained therein.
 13. The method according to claim 12, furthercomprising the step of storing the sample after performing theincubating step and before performing the separating step.
 14. Themethod according to claim 12, wherein the reagent further contains 1.00mg/l of dye.
 15. The method according to claim 12, wherein the pathogensare spirochetes.